Crossovers direct the accurate segregation of meiotic chromosomes. Defects in meiotic recombination lead to aberrant chromosome segregation. In severe cases, this results in sterility. When only one chromosome pair is affected, loss of meiotic recombination can give rise to monosomies and trisomies. It is estimated that 25-30% of human conceptions result in spontaneous termination due to these defects. Although extensive research, primarily in S. cerevisiae, has elucidated many details of the meiotic recombination pathway, we still do not know how recombination intermediates are processed to generate crossovers. It has become evident that the processes that actually generate crossovers vary widely in different organisms, so it is important to develop additional models. In Drosophila melanogaster, several conserved proteins whose homologs have well-characterized functions in other pathways are used in novel ways to generate meiotic crossovers. To understand how crossovers are generated in Drosophila, we will combine genetic and molecular analyses of recombination products from various mutants with biochemical studies of the proteins involved. We will first recover intragenic recombination events from mismatch repair mutants. We will use multiple molecular markers to determine the contribution of each homolog to recombination products. This will allow us to distinguish between different models for recombination. We will then repeat these experiments in mutants that are unable to generate crossovers, including mei-9 and rec. MEI-9 is a Drosophila nucleotide excision repair endonuclease and is required to generate crossovers and conduct mismatch repair in meiosis. REC, the Drosophila ortholog of MCM8, is also required to generate meiotic crossovers. Analysis of recombination events in these mutants will test hypotheses concerning the functions of these proteins in generating crossovers. We will also test the hypothesis that MEI-9 and partner proteins cut Holliday junctions by purifying complexes from insect cells and assaying activities on model synthetic substrates. The work proposed here will provide a greater understanding of meiotic recombination mechanisms in a model metazoan and will allow comparison of recombination mechanisms in different organisms.